Method and apparatus for purifying and desalting biological samples

ABSTRACT

The subject invention provides a sample processing technique for purifying a biological or chemical sample. The invention is particularly well-suited to prepare a sample for mass spectrometry. The process is to be performed in an article having at least one well, in which the surface of the well is at least partially hydrophobic and/or modified with bio-specific ligands. Targeted solutes, such as salts or small molecule contaminants, can be removed from a solution to allow for a purified solution of a desired type of solute, such as peptides and/or proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/844,777, now allowed, filed May 13, 2004 and claims priority of U.S.Provisional Patent Application No. 60/469,986, filed May 13, 2003; U.S.Provisional Patent Application No. 60/470,021, filed May 13, 2003; U.S.Provisional Patent Application No. 60/538,913, filed Jan. 23, 2004; andU.S. Provisional Patent Application No. 60/564,927, filed Apr. 23, 2004,all of which are hereby incorporated by reference.

FIELD OF INVENTION

This invention relates to methods and apparatuses for processingbiological and chemical samples.

BACKGROUND OF THE INVENTION

Conventional techniques exist for conducting mass spectrometric analysisof large molecules using MALDI (matrix-assisted laser desorption andionization) plates. Typically with these techniques, liquid solutions(including e.g., peptide, protein, and energy absorbing matrix) areinitially introduced to pre-defined target sites on a MALDI plate. Sincethe diameter of the target sites are generally small and often denselypacked, small (e.g., 1-5 microliter) droplets of the liquid solutionsare disposed onto the plate target sites to achieve proper sampleplacement and to avoid sample overlap between target sites. Oncedisposed, the liquid samples are evaporated, with matrix crystalconglomerate containing analyte molecules (e.g., peptides and proteins)remaining on the target sites having favorable characteristics for theMALDI process and mass spectrometric analysis. Where larger conglomeratesamples are desired, serial liquid sample placement and evaporation hasbeen used to iteratively build-up a conglomerate.

Prior to placement on the MALDI plate, the liquid solution is typicallypurified and desalted. Such purification and desalting is achieved inthe prior art with columns, pipette tips or multi-well filter platesbeing packed with C18 media or other chromatography media. Examples ofcolumns packed with C18 media include Pierce PepClean™ C18 Spin Columnsand 3M Empore™ Extraction Disk Cartridge. Examples of pipette tipspacked with C18 media include Millipore ZipTip® Pipette Tips and VarianOMIX Pipette Tips C18. Examples of multi-well filter plates packed withC18 media include Millipore Zip Plate Micro-SPE Plate and 3M Empore™96-Well Extraction Disk and Plate.

For exemplary purposes, a common procedure for purifying and desaltingliquid solution samples using one of the above-identified devicesincludes initially wetting the C18 media with a buffer containing anorganic solvent, such as methanol or acetonitrile, and thereafter,equilibrating the C18 media with an equilibration buffer. The liquidsolution samples are then flowed through the C18 media; it is expectedthat peptides and proteins of the sample be retained in the C18 mediaduring this step. Thereafter, the C18 media is washed with a washingbuffer, and, again, the peptides and proteins are expected to beretained in the media during this step. Finally, a flow elution bufferis passed through the C18 media which disassociates the peptides andproteins from the media, and it is expected that the peptides andproteins flow out with the elution buffer in this step.

A significant portion of the peptides and proteins in the samplesolution may flow through the C18 media or other chromatography media inthe prior art devices without binding to the C18 or other media. As aresult, recovery of the peptides and proteins may be poor, particularlywhere the sample concentration is low.

SUMMARY OF THE INVENTION

The subject invention provides a sample processing technique forpurifying a biological or chemical sample. The invention is particularlywell-suited to prepare a sample for mass spectrometry, such as preparinga sample for use with a MALDI plate. The process is to be performed inan article having at least one well, in which the surface of the well isat least partially hydrophobic and/or modified with bio-specificligands. With the process of the subject invention, a sample solution isadded to the well, with the solution including two types of solutes: afirst type of solutes (such as peptides and proteins) which are able tobind tightly to the well surface through hydrophobic interactions orthrough interactions with the bio-specific ligands immobilized on thewell surface, and a second type of solutes (such as salts or smallmolecule contaminants) which are not able to bind tightly to the wellsurface. Once added, the sample solution is dried in the well, leavingall of the solutes deposited on the well surface. A buffer, which is notable to disassociate the first type of solutes from the well surface butis able to disassociate the second type of solutes from the wellsurface, is added to the well. Consequently, the second type of solutesis dissolved into the first buffer, while the first type of solutesremains bound to the well surface. The first buffer, now containing thesecond type of solutes, is removed from the well, leaving the first typeof solutes bound to the well surface. The first buffer can be seriallyadded and removed to maximally remove the second type of solutes.Thereafter, a second buffer, which is able to disassociate the firsttype of solutes from the well surface, is added to the well. The firsttype of solutes is dissolved into the second buffer, thus, providing apurified solution of the first type of solutes. Optionally, the secondbuffer may be allowed to evaporate, thereby, increasing itsconcentration.

The process of the subject invention can be practiced in a column,pipette or multi-well plate, with the purified solution being laterapplied to a target plate or other desired analysis device.Additionally, the process of the subject invention can be practiced witha target support plate which can be releaseably secured to a targetdevice, such as a MALDI plate. With a target support plate, the liquidsample can be purified in the target support plate and on the device,and, then, evaporated to allow a conglomerate to be directly formed onthe target plate with no transference of the liquid sample beingrequired.

These and other features of the invention will be better understoodthrough a study of the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 1(b) and 1(c) are schematics, of a multi-well plate, a column,and a pipette usable with the subject invention.

FIGS. 2( a)-(f) show the process of the subject invention beingpracticed in a multi-well plate.

FIGS. 3( a) and (b) schematically show a target support plate and targetdevice usable with the subject invention.

FIGS. 4-7 shows various target support plate and target deviceassemblies, wherein FIG. 5 is an enlarged view of Section 5 in FIG. 4.

FIGS. 8( a)-(j) show the process of the subject invention beingpracticed with a target support plate and target device.

FIGS. 9-15 show various mass spectra.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a new process for purifying a biological or chemicalsample. The process is particularly well-suited for preparing liquidsamples for mass spectrometry, such as preparing a sample for use with aMALDI plate. As will be described below, the process can be practicedremotely from the device intended to analyze the sample or practiced incontiguous contact therewith.

With reference to FIGS. 1 and 2( a)-(f), a multi-well plate 11 is shownhaving one or more wells 12 defined therein as is known in the art. Atleast one of the wells 12 is formed with a surface that is hydrophobicand/or modified with bio-specific ligands (such as immobilized metal ionaffinity chromatography (IMAC) matrices for phosphorylatedpeptides/proteins or poly(histidine) fused peptides/proteins, biotinaffinity matrices for biotinylated peptide/proteins, and thiol-disulfideexchange chromatography matrices for glutathione S-transferase (GST)fused peptides/proteins). It is to be understood that the process of thesubject invention can be practiced with structures other than amulti-well plate, including, but not limited to, a target support plate(as described below), a column 11 b (FIG. 1( b)) and a pipette 11 c(FIG. 1( c)). With any structure, it is desired that a well be providedwithin the device in which a surface that comes into contact with theliquid sample be hydrophobic and/or modified with bio-specific ligands.The entire well surface need not be hydrophobic and/or modified withbio-specific ligands. The well surface can be inherently hydrophobic ortreated to be hydrophobic (e.g., by coating with alkyl silanes orhydrophobic polymers).

As shown in FIGS. 2( a)-(f), the subject invention includes sequentialsteps for purifying a liquid sample. The liquid sample may be a knownliquid sample used in preparing analytes for the MALDI process or othermass spectrometric processes (such as liquid samples which containtryptic digest products). Specifically, the process seeks to eliminatesalts and other contaminants from a sample solution, while maintainingpeptides and proteins therein at the highest possible level. Withreference to FIG. 2( a), a liquid sample solution 14 is introduced intoone or more wells 12. The solution 14 contains two types of solutesdesignated with the reference numerals 15 and 16 (the black solidcircles represent the first type of solutes 15 while the open trianglesrepresent the second type of solutes 16). The first type of solutes 15are able to bind tightly to the surfaces of the wells 12 throughhydrophobic interactions or through interactions with the bio-specificligands immobilized on the well surfaces. The first type of solutes 15may include peptides and proteins. It should be noted that when peptidesand proteins are in proximity to a hydrophobic surface in aqueoussolutions, they will be adsorbed to the hydrophobic surface. The secondtype of solutes 16 are not able to bind tightly to the well surfaces andmay be salts or small molecule contaminants.

Once the solution is deposited, the solution 14 is evaporated or driedoff leaving the solutes 15 and 16 deposited on the well surfaces, asshown in FIG. 2( b). Thereafter, a first buffer 17, which is not able todisassociate the first type of solutes 15 from the well surfaces but isable to disassociate the second type of solutes 16 from the wellsurfaces, is added to the wells 12 (FIG. 2( c)). The first buffer 17preferably is primarily formed of water. As shown in FIG. 2( c), thesecond type of solutes 16 dissolve in the first buffer 17, while thefirst type of solutes 15 remain bound to the well surfaces. Withreference to FIG. 2( d), the first buffer 17, containing the second typeof solutes 16, is removed from the wells 12, leaving the first type ofsolutes 15 bound to the well surfaces. The first buffer 17 can beserially added and removed to maximally remove the second type ofsolutes 16. Thereafter, a second buffer 18 is added to the wells 12. Thesecond buffer 18 is able to disassociate the first type of solutes 15from the well surfaces. Preferably, the second buffer 18 contains anorganic solvent such as acetonitrile or methanol, and, more preferably,is primarily acetonitrile. Additionally. the second buffer 18 mayinclude an energy absorbing matrix such as α-cyano-4-hydroxy cinnamicacid, 3,5-dimethoxy-4-hydroxy cinnamic acid, or 2,5-dihydroxybenzoicacid. With the second buffer 18, the first type of solutes 15 aredissolved into the second buffer 18 providing a purified solution of thefirst type of solutes 15 (FIG. 2( e)). Optionally, and as shown in FIG.2( f), a higher concentration of the first type of solutes 15 can beobtained by allowing the second buffer 18 to evaporate.

As described above, the subject application can be used with variousdevices which define one or more of the wells 12. The wells 12 may bedefined wholly by one component (e.g., a multi-well plate) or by acombination of two or more components, such as with a target supportplate secured to a target device. With reference to FIGS. 3( a)-(b), atarget support plate 21 is shown having one or more columns 22 extendingbetween, and through, top and bottom surfaces 30 and 31. The targetsupport plate 21 is releasably securable to a target device 23, such asa MALDI plate or other mass spectrometry plate having one or morecollection sites (e.g., a SELDI (Surface Enhanced LaserDesorption/Ionization) plate, or a DIOS (Desorption/Ionization on PorousSilicon) plate). The columns 22 and the target device 23 collectivelydefine fluid-containing wells. Preferably, the target support plate 21is formed of an elastomeric material which can be releasably secured tothe target device 23. It is preferred that the elastomeric materialinclude a silicon polymer, and more preferably, includepoly(dimethyl)siloxane (PDMS). With an elastomeric material, van derWaals interactions between the surface molecules of the target supportplate 21 and the target device 23 provide for a releasable securement.The target support plate 21 can be pressed onto the target device 23 forsecurement and removed therefrom by peeling. It is further preferredthat the body of the target support plate 21 be wholly formed of theelastomeric material, more preferably being wholly formed of PDMS. Theelastomeric and hydrophobic natures of PDMS allow for a tight bond to beformed between the target support plate 21 and the target device 23.With the target support plate 21 being only partially formed of theelastomeric material, remaining portions may be formed of rigid plasticor other material which will impart favorable characteristics to thewalls of the columns 22. The securement is preferably of sufficientintegrity to prevent cross-contamination between the columns 22 alongthe target support plate 21/target device 23 interface. Optionally, thewalls of the columns 22 may be modified with bio-specific ligands.

Other forms of the target support plate 21 may be used which allow forreleasable securement with the target device 23. The target supportplate 21 may rely on adhesive, an elastomeric gasket, and/or areleasable mechanical fixation to allow for releasable securementbetween the target support plate 21 and a target device 23. Withreference to FIGS. 4 and 5, a mechanical fixation is disclosed, whereina mechanical locking member 38 may be provided which protrudes from thebottom surface 37 to at least partially bound the target device 23. Thelocking member 38 includes an upstanding support member 40 and atransverse member 42. The upstanding support member 40 and thetransverse member 42 are formed such that a portion of the target device23 is interposed between an engagement surface 44, defined on thetransverse member 42, and the bottom surface 37. The transverse member42 may also include a rearwardly, extending protruding member 46. Thetarget device 23 can be “snapped” into releasable securement with thelocking member 38 deflecting and returning to the position shown inFIGS. 4 and 5. Removal of the target device can be achieved by rearwarddisplacement of the protruding member 46 resulting in moment beingapplied about the upstanding support member 40, deflection of thelocking member 38, and separation of the engagement surface 44 from thetarget device 23. As can be appreciated, the strength of the holdingforce applied to the target device 23, as well as the difficulty ofsecurement and removal of the target device 23, will be a function ofthe strength of the locking member 38, and the extent to which thelocking member 38 bounds the target device 23.

As shown in FIG. 6, adhesive 48 may be used to releasably secure thetarget device 23 to the target support plate 21. Any suitable adhesivemay be used which will allow for release of the target support plate 21,yet provide sufficient holding force to the target support plate 21 toallow for preparation of collection sites 34.

As shown in FIG. 7, an elastomeric gasket 50 may be interposed betweenthe target device 23 and the target support plate 21 to providereleasable securement therebetween. In the same manner as describedabove with the body of the target support plate 21 being formed of anelastomeric material, the elastomeric gasket 50 provides releasableadhesion. This adhesion may be achieved by van der Waals interactions.Preferably, the elastomeric material of the gasket 50 includes siliconpolymer, and more preferably, includes poly(dimethyl)siloxane (PDMS).The elastomeric material may also be doped with other polymers tocustomize its physical properties. It is further preferred that theelastomeric gasket 50 be wholly formed of PDMS. Apertures 52 shall beformed in the gasket 50 as required to expose the intended collectionssites 34. It is preferred that the apertures 52 each have a diameterthat is greater than, or equal to, that of the open bottom ends of thecolumns 22.

As will be understood by those skilled in the art, regardless of themanner by which releasable securement is achieved, it is desired thatsufficient sealing be provided along the interface between the targetsupport plate 21 and the target device 23 to prevent cross-contaminationof any liquid samples contained in the columns 22. The sealing should beat least fluid-tight. In addition, the level of strength of thereleasable securement must be considered in view of any processing stepsthe assembly is to be subjected to. Adhesive and elastomeric sealingwill generally provide a weaker holding force than a mechanical fixationand may be used with smaller volume liquid samples and/or lighter targetdevices; whereas, a mechanical fixation may be used with larger liquidsamples and/or heavier target devices. This is particularly so where theassembly is intended to be centrifuged or otherwise transported togetherwith releasable securement being maintained. On the other hand, thetarget device 23 should be detached without damage thereto. The variousforms of releasable securement can be used in varying combinations (forexample, adhesive may be used in combination with mechanical fixation).

It is preferred that the target support plate 21 be wholly formed fromPDMS. PDMS is inherently hydrophobic, and as such, by wholly forming thetarget support plate 21 of PDMS, the walls of the columns 22 will beinherently hydrophobic.

With reference to FIGS. 8( a)-(j), the process of the subject inventionis shown in conjunction with the use of the target support plate21/target device 23 combination. Initially, as shown in FIGS. 8( a) and(b), the target support plate 21 is secured to the target device 23.Thereafter, a liquid sample solution 24 (such as a solution containingtryptic digest products) is placed into the columns 22 (FIG. 8( c))which includes two types of solutes 25 and 26: the first type of solutes25 (represented by solid circles) are able to bind tightly to the columnsurfaces through hydrophobic interactions or through interactions withthe bio-specific ligands immobilized on the column surfaces (e.g.,peptides and proteins), while the second type of solutes 26 (representedby open triangles) are not able to bind tightly to the column surfaces,e.g., salts or small molecule contaminants. Upon drying or evaporatingthe sample solution 24 (FIG. 8( d)), the solutes 25 and 26 are depositedon the column surfaces and possibly to some extent on the target device23. Because of adsorptive attraction, the first type of solutes 25 willtend to be deposited on the walls of the columns 22, rather than on thesurface or collection site 34 of the target device 23. A first buffer 27is next added which is not able to disassociate the first type ofsolutes 25 from the surfaces of the columns 22 but is able todisassociate the second type of solutes 26 from the column surfaces. Asa result, the second type of solutes 26 are dissolved into the firstbuffer 27, while the first type of solutes 25 remain bound to the columnsurfaces (FIG. 8( e)). The first buffer 24, containing the dissolvedsecond type of solutes 26, is removed from the columns 22, leaving thefirst type of solutes 25 bound to the column surfaces (FIG. 8( f)). Thesteps of adding and removing the first buffer 27 can be repeatedlyconducted to maximally remove the second type of solutes 26.Subsequently, a second buffer 28, as shown in FIG. 8( g), is added tothe columns 22, which is able to disassociate the first type of solutes25 from the column surfaces. As a result, the first type of solutes 25is dissolved into the second buffer 28, thereby providing a purifiedsolution of the first type of solutes 25. The second buffer 28 can beevaporated to increase the concentration of the resulting liquid (FIG.8( h)). Complete evaporation of the second buffer 28 results in thedeposition of the first type of solutes 25 onto the target device 23within the areas at the bottom of the respective columns 22 (FIG. 8(i)). The deposition sites coincide with the collection sites 34 on thetarget device 23. For analysis purposes, the target support plate 21 isremoved from the target device 23, for example by peeling (FIG. 8( j)).The deposited first type of solutes 25 form sample spots which can beused in further analysis, such as with mass spectrometry, e.g., MALDImass spectrometry analysis.

As with the method described above, the first buffer 27 is preferablyprimarily of water, and the second buffer 28 contains an organicsolvent, such as acetonitrile or methanol, and, more preferably,primarily acetonitrile. Additionally. the second buffer 28 may includean energy absorbing matrix such as α-cyano-4-hydroxy cinnamic acid,3,5-dimethoxy-4-hydroxy cinnamic acid, or 2,5-dihydroxybenzoic acid.

With the process of the subject invention, purer samples can be obtainedthan in the prior art which result in better analysis. FIGS. 9-12 show acomparison between the same sample solutions having been processed usingpipette tips with C18 media versus the method described above inconjunction with FIG. 8. FIGS. 9( a), 10(a), 11(a) and 12(a) are massspectra obtained from sample solutions prepared by pipette tips with C18media, while FIGS. 9( b), 10(b), 11(b) and 12(b) were prepared using themethod associated with FIG. 8 of the subject application. The massspectra of FIG. 9 were obtained for a sample solution (50 μL) containing100 nM peptide standards; the mass spectra of FIG. 10 were obtained fora sample solution (50 μL) containing 50 nM peptide standards; the massspectra of FIG. 11 were obtained for a sample solution (50 μL)containing 20 nM peptide standards; and the mass spectra of FIG. 12 wereobtained for a sample solution (50 μL) containing 10 nM peptidestandards. The arrows below the x-axis in each of the mass spectraindicate the expected peak positions of the six standard peptides (Humanbradykinin fragment 1-7, M.W.=757.4; Human angiotensin II, M.W.=1046.5;Synthetic peptide P₁₄R, M.W.=1533.9; Human ACTH fragment 18-39,M.W.=2465.2; Bovine insulin oxidized B chain, M.W.=3494.7; Bovineinsulin, M.W.=5734.5), while a “X” indicates that a matching peak isfound in the spectrum. It must be noted that some of the peaks in thelow molecular weight region are contributed by the MALDI matrixα-cyano-4-hydroxy cinnamic acid (CHCA). As will be noted, more matchingpeaks were detected by the subject invention than with the conventionaltips with C18 media.

FIG. 13-15 show a similar comparison, where FIG. 13( a), 14(a) and 15(a)were obtained by using pipette tips with C18 media, while FIGS. 13( b),14(b) and 15(b) were obtained using the subject invention. The massspectra in FIG. 13 were obtained for a sample solution (50 μL)containing 50 nM Bovine Serum Albumin (BSA) tryptic digest, the massspectra in FIG. 14 were obtained for a sample solution (50 μL)containing 20 nM BSA tryptic digest, and the mass spectra in FIG. 15were obtained for a sample solution (50 μL) containing 10 nM BSA trypticdigest. As can be seen from FIGS. 13-15, more peptide peaks from the BSAtryptic digest were resolved by the subject invention than with theconventional tips with C18 media, indicating a better recovery ofpeptides from the purification process.

Various changes and modifications can be made in the present invention.It is intended that all such changes and modifications come within thescope of the invention as set forth in the following claims.

1. A process for purifying a biological or chemical sample, said processcomprising: providing an article having a well with a surface which isat least partially modified with bio-specific ligands; depositing aliquid sample in said well; evaporating said liquid sample with a firstclass of solutes in said sample tightly binding to said surface, and asecond class of solutes in said sample not tightly binding to saidsurface; depositing a first buffer in said well, said first bufferdisassociating said second class of solutes from said surface; removingsaid first buffer and said disassociated second class of solutes fromsaid well; and depositing a second buffer in said well after removingsaid first buffer, said second buffer disassociating said first class ofsolutes from said surface.
 2. A process as in claim 1, wherein saidarticle is a multi-well plate.
 3. A process as in claim 1, wherein saidarticle is a column.
 4. A process as in claim 1, wherein said article isa pipette.
 5. A process as in claim 1, wherein said article includes atarget support plate having spaced-apart top and bottom surfaces, atleast one column extending between, and through, said top and bottomsurfaces, and a target device releasably secured to said target supportplate, said target device having at least one collection site, saidcolumn registering with said collection site, said column and saidcollection site collectively defining said well.
 6. A process as inclaim 5, wherein said target device is releasably secured to said targetsupport plate.
 7. A process as in claim 5, wherein at least the bottomsurface of said target support plate is formed of an elastomericmaterial releasably adhered to said target device.
 8. A process as inclaim 7, wherein said elastomeric material includes a silicon polymer.9. A process as in claim 7, wherein said elastomeric material includespoly(dimethyl)siloxane.
 10. A process as in claim 7, wherein said targetsupport plate is wholly formed of said elastomeric material.
 11. Aprocess as in claim 5, wherein said target device is a mass spectrometryplate.
 12. A process as in claim 1, wherein said liquid sample includestryptic digest products.
 13. A process as in claim 1, wherein said firstclass of solutes includes peptides.
 14. A process as in claim 1, whereinsaid first class of solutes includes proteins.
 15. A process as in claim1, wherein said second class of solutes includes salts.
 16. A process asin claim 1, wherein said second class of solutes includes small moleculecontaminants.
 17. A process as in claim 1, wherein said first bufferincludes water.
 18. A process as in claim 1, wherein said second bufferincludes an organic solvent.
 19. A process as in claim 1, wherein saidsecond buffer includes an energy absorbing matrix.
 20. A process as inclaim 1, wherein said bio-specific ligands may be one or more selectedfrom the group consisting of immobilized metal ion affinitychromatography (IMAC) matrices for phosphorylated peptides/proteins orpoly(histidine) fused peptides/proteins, biotin affinity matrices forbiotinylated peptide/proteins, and thiol-disulfide exchangechromatography matrices for glutathione S-transferase (GST) fusedpeptides/proteins.